1. Field of the Invention
The present invention relates to biocompatible hyaluronan-containing materials and uses thereof.
2. Description of the Related Art
TNF-Stimulated Gene 6 (TSG-6)
TNF-stimulated gene 6 (TSG-6) encodes a glycoprotein of ca. 35 kDa that is commonly referred to as TSG-6 protein (Lee et al., 1990 and 1992; and Wisniewski et al., 2004). Expression and function of TSG-6 have been associated with inflammation and fertility.
TSG-6 protein consists of two domains, the N-terminal link module and the C-terminal CUB domain. The N-terminal domain of TSG-6, a so-called link module (Kohda et al., 1996), identifies TSG-6 as a hyaluronan (HA) binding protein. All known proteins sharing this domain are hyaladherins, i.e., HA-binding proteins (Iozzo et al., 1996; Knudson et al., 1993; and Toole, 1990). Not surprisingly, TSG-6 has been shown to bind to HA in solution (Lee et al., 1992; and Kohda et al., 1992) and to immobilized HA (Kahmann et al., 2000; Mahoney et al., 2001; Parkar et al., 1998; and Wisniewski et al., 2005). TSG-6 is the only known HA-binding protein that contains a CUB domain, which may account for some of its unique properties. The 3D structure of the link module of TSG-6 has been solved and many structural details of its interaction with HA have been investigated (Kohda et al., 1996; Kahmann et al., 2000; and Mahoney et al., 2001). The laboratory of the present inventors have previously reported that TSG-6 formed complexes with HA covalently attached to a solid substrate and that these HA-TSG-6 complexes were resistant to dissociation with guanidine HCl, guanidine HCl containing lauryl sulfobetain, SDS plus 2-mercaptoethanol, and dilute NaOH, consistent with the formation of a covalent bond (Wisniewski et al., 2005). The isolated link module is likewise competent to form these tight complexes, indicating that the CUB domain of TSG-6 is not required for this interaction with HA. The CUB domain, a modular unit that is widely shared by numerous proteins, is thought to be a protein-protein and protein-carbohydrate interaction domain (Bork et al., 1993; and Topfer-Petersen et al., 1998).
TSG-6 is a protein whose expression is induced by the pro-inflammatory cytokines TNF-α, IL-1 and IL-17. TSG-6 protein has shown potent anti-inflammatory and tissue-protective activities in experimental models of acute and chronic inflammation. TSG-6 protein has been particularly impressive in ameliorating experimental arthritis, which has been shown in three different models of experimental arthritis using either recombinant TSG-6 or endogenous expression of TSG-6 in two different models of transgenic mice. In addition, TSG-6-deficient mice developed aggravated arthritis and cartilage destruction.
TSG-6 and the ubiquitous plasma protein inter-α-inhibitor (IαI) form a biochemical pathway to permanently modify hyaluronan (HA), a glycosaminoglycan abundant in many tissues. HA is particularly prominent in joints where it serves both as a major structural component of cartilage and, being present at high concentration in synovial fluid, a viscoelastic lubricant. TSG-6 interacts with IαI in the absence of any other factors and serves as acceptor of one heavy chain of IαI, forming a stable TSG-6-HC complex that serves as a stable intermediate for the transfer of HCs to hyaluronan. The resulting HA-HC complexes are stable and have been found in the synovial fluids of patients with rheumatoid arthritis and osteoarthritis (Kida et al., 1999).
IαI is a protein-polysaccharide complex of unique structure, consisting of three polypeptide chains linked by a glycosaminoglycan (GAG) bridge (Enghild et al., 1991; and Salier et al., 1996). The smallest of the three polypeptides, the serine protease inhibitor bikunin, carries a single chondroitin-4-sulfate chain attached via a classical proteoglycan linkage group, while the two closely homologous heavy chains (HC) 1 and 2 are linked to hydroxyl groups of the chondroitin sulfate via ester bonds formed by their C-terminal aspartic acid residues (Enghild et al., 1989, 1991 and 1993). Purified TSG-6, in the absence of HA or any added factors, interacts with IαI resulting in the transfer of one HC from IαI to TSG-6 (Mukhopadhyay et al., 2004; Sanggaard et al., 2005 and 2006; and Wisniewski et al., 1994).
It is intriguing that the HCs of IαI are also found in stable complexes with HA, and have been named serum-derived HA-associated protein (SHAP) (Huang et al., 1993). As in IαI, the HCs are coupled to HA by an ester bond formed by its C-terminal aspartic acid residue (Zhao et al., 1995). The formation of HA-HC complexes has been described both in the absence (Huang et al., 1993) or in the presence of added TSG-6 (Mukhopadhyay et al., 2004; and Rugg et al., 2005).
The HCs of IαI have also been reported to have anti-inflammatory effects in their own right, suggested to be mediated by inhibition of complement activation by circulating immune complexes. Activation of complement in the vascular compartment has been associated with autoimmune and inflammatory conditions, e.g., systemic lupus erythematosus, resulting in platelet damage and fibrin deposition, both in turn causing perpetuation of inflammation.
TSG-6 has been associated with various forms of arthritis (Bayliss et al., 2001; and Wisniewski et al., 1993) and it has been demonstrated by several investigators to exert anti-inflammatory and chondroprotective effects in murine models of acute inflammation and autoimmune arthritis (Bardos et al., 2001; Getting et al., 2002; Glant et al., 2002; Mindrescu et al., 2000 and 2002; and Wisniewski et al., 1996). TSG-6 and IαI play an essential role in female fertility, and both TSG-6- and IαI-deficient female mice are essentially infertile because their ovaries fail to form the HA-rich protective cumulus surrounding oocytes during ovulation (Fulop et al., 2003; Sato et al., 2001; and Zhuo et al., 2001). The drop of IαI concentrations in human plasma during sepsis and the beneficial effects of exogenous IαI in experimental models of sepsis point to a significant role of IαI in human disease (Baek et al., 2003; Balduyck et al., 2000; Lim et al., 2003; Opal et al., 2007; Wu et al., 2004; and Yang et al., 2002). Thus, IαI is considered a prognostic marker for the outcome of septic shock in humans, with low concentrations of IαI predicting mortality. In experimental sepsis, IαI has been shown to increase survival. Bikunin, the protease inhibitory chain of IαI, is also found as a free polypeptide in urine and is therefore also known as urinary trypsin inhibitor (UTI). Bikunin/UTI has been described as having anti-inflammatory and anti-metastatic effects in a range of experimental systems (Kobayashi et al., 2003 and 2006; and Pugia et al., 2005).
IαI also interacts with pentraxin 3 (PTX3), a pathogen-associated molecular pattern receptor. This connection ties IαI firmly to the innate immune response. PTX3 is essential for efficient innate immunity to Aspergillus fumigatus infections in the mouse. PTX3 is an activator of complement, and its function in the innate immune system may be related to this ability. IαI binds to PTX3 and may modulate the activity of PTX3 by its ability to modulate complement activation.
TSG-6, IαI, hyaluronan and PTX3 are collectively responsible for the stability of the expanding cumulus-oocyte complex during ovulation, and are therefore essential for female fertility. These components were also recently reported US2070231401 to occur in extracts of amniotic material with anti-inflammatory properties.
Hyaluronan
Hyaluronan is a carbohydrate polymer (polysaccharide), which is normally found in the matrix surrounding cells in vertebrate animals, and is a major component of the vitreous of the eye and the synovial fluid of the joint. Hyaluronan is synthesized by a cell surface enzyme, and extruded directly into the extracellular matrix. Although some hyaluronan is degraded locally, most is transported through the lymph and degraded in the lymph nodes, with most of the remaining amount being cleared rapidly from the blood and degraded by liver endothelial cells. Tissue hyaluronan has a rapid turnover, with approximately one-third of the total being degraded and replaced each day.
Commercial preparations of hyaluronan are isolated from rooster comb or from the culture medium of certain bacteria that are capable of synthesizing the polysaccharide. Hyaluronan contains two different sugar units, which alternate in the polymer, forming a linear chain. The number of sugar units in a single chain of the natural material can reach at least 40,000, which corresponds to a molecular weight of 8,000,000. Commercial preparations of hyaluronan are usually lower in molecular weight, due to degradation during isolation and purification, and are polydisperse (i.e., contain a range of molecular weights). In referring to the molecular weight, an average molecular weight is cited.
A large number of different biomaterials and therapeutic products containing hyaluronan have been proposed for use, and some have been commercialized. Pure hyaluronan is non-immunogenic and has excellent biocompatibility.
References for recent authoritative reviews of the chemistry, biology, and medical applications of hyaluronan are provided below (Lap{hacek over (c)}ik et al., 1998; Balazs, 2004; Asari, 2004; Miller and Avila, 2004; Shu and Prestwich, 2004; Cowman and Matsuoka, 2005; Morra, 2005; Brekke and Thacker, 2006; Stern et al., 2006; Kogan et al., 2007).
Hyaluronan to be Used in Soluble Unmodified Form
The medical applications of soluble unmodified hyaluronan include uses that depend primarily on the physical properties (viscosity, elasticity, osmotic pressure, etc.), and uses that depend wholly or in part on binding of hyaluronan to cell surface receptors. The molecular weight of the hyaluronan, and the concentration of hyaluronan in solutions, are important considerations in both the physical properties and the cell receptor interactions of hyaluronan.
Physical Properties Solutions of high molecular weight (ca. 400,000 to 6,000,000) hyaluronan are notable for their high viscosity and elastic character, both of these depending also on the concentration and the frequency of deformation (shear rate). Hyaluronan of lower molecular weight has much lower viscoelasticity. Hyaluronan solutions have a high osmotic pressure and contribute to tissue hydration.
Based primarily on the viscoelastic properties, solutions of high molecular weight hyaluronan have been used extensively as tissue protectants and manipulators (viscous tools) in ophthalmic applications, most notably cataract extraction coupled with intraocular lens implantation. Solutions of high molecular weight hyaluronan are also widely used for pain relief in treatment of osteoarthritis of the knee.
Applications based primarily on the hydration properties include protective eye drops for treatment of dry eye, and skin moisturizers for use in cosmetics.
Applications that depend strongly on both hydration and viscous properties include 1) tissue protectants for use in minimizing tissue abrasion, post-surgical adhesion formation, or loss of natural protectant layers in peritoneal dialysis, and 2) wound protectant and healing aids.
Solutions of high molecular weight hyaluronan at sufficiently high concentration to have significant crowding and spatial overlap of the polymer chains have the property of slowing the diffusion of other co-dissolved molecules. Hyaluronan has been suggested as an adjuvant for slowed diffusion of therapeutic agents (e.g, for use in joints, wounds, ulcers, burns, etc.).
Cell Surface Receptor-Mediated Properties. Cell surface receptor interactions may play a role in several of the above applications. It is known that hyaluronan molecular weight influences the cell surface receptor interactions. High molecular weight hyaluronan, bound to the receptors, is characteristic of the healthy physiological environment. In several pathological conditions, hyaluronan is degraded or synthesized at a lower molecular weight. Hyaluronan with a molecular weight of less than approximately 200,000 binds to cell surface receptors in an altered manner, resulting in signaling that leads to expression of genes for proteins that mediate the inflammatory response. Furthermore, small oligosaccharides (containing less than about 50 sugars in a chain) of hyaluronan have still different cell signaling properties. Oligosaccharides can induce cell death in tumor cells, make tumor cells more sensitive to chemotherapy drugs, induce blood vessel growth, rescue cells from inflammation, and other seemingly conflicting activities. Each of these biological activities appears to depend on a particular chain size. The mechanisms for these effects, and the reasons for the molecular weight dependence, remain to be explained in detail. Products under development include hyaluronan fragments of specific sizes.
Noncovalent Complexes of Unmodified Hyaluronan with Other Agents
A gel-like solution of the Fe3+ salt of hyaluronan was developed for use as an antiadhesion material in pelvic surgery, but was withdrawn. A Cu2+ salt of hyaluronan was suggested to aid cell adhesion (Barbucci et al., 2000). Ca2+ was used to enhance the effect of hyaluronan in slowing the diffusion of the drug doxycycline. A mixture of hyaluronan with KI3 (which may be weakly complexed together) has been proposed for use in wound healing (Frankova et al., 2006).
Complexes of hyaluronan with positively charged drug molecules, stabilized by both electrostatic and hydrophobic interactions, were suggested for use in drug delivery (Santos et al., 2007).
Complexes of the negatively charged hyaluronan with positively charged polymers (chitosan, polylysine, etc.) are generally insoluble or slowly soluble in physiological media. The polyelectrolyte complexes have been prepared as fibers, microspheres, surface coatings, and multilayer composite materials. The layers can also be crosslinked into microshells (Lee et al., 2007a). The intended uses of these materials include anti-coagulant blood vessel wall coatings to combat re-stenosis (Thierry et al., 2003a), cell culture substrates, tissue engineering scaffolds, and controlled drug release agents. Another tissue engineering matrix composed of hyaluronan-chitosan fibers embedded in a more soluble mixture of hyaluronan and chitosan has been proposed. Hyaluronan has also been complexed with polypyrrole to produce an electrically conducting material to stimulate nerve tissue regrowth. In that case, the intended role of the hyaluronan is slow degradation to release bioactive oligosaccharides.
Insoluble or slowly soluble electrospun fibers of hyaluronan, hyaluronan/gelatin mixtures, or hyaluronan crosslinked by poly(ethylene glycol) have been prepared for use in tissue engineering (Um et al., 2004; Li et al., 2006; Ji et al., 2006a, 2006b).
Hyaluronan can be infused into a porous three-dimensional bone graft scaffold composed of polylactic acid, to facilitate cellular infiltration.
Soluble Chemical Derivatives of Hyaluronan
Hyaluronan can be derivatized by a number of procedures, examples of which are described in the publications of Vercruysse and Prestwich, 1998, Prestwich et al., 1998; Luo and Prestwich, 2001, and Shu and Prestwich, 2004.
The protein superoxide dismutase has been chemically attached to hyaluronan for use as an anti-inflammatory agent (Sakurai et al., 1997).
Chemical attachment of small molecule therapeutic agents such as                doxorubicin or other antiproliferatives (Luo and Prestwich, 1999; Rosato et al., 2006),        methylprednisolone or other steroid esters (Taglienti et al, 2005),        diclofenac or other non-steroidal anti-inflammatory agents,        bupivacaine or other analgesics,        nitric oxide for the inflammatory phase of wound healing (DiMeo et al, 2006),        other therapeutic moleculesmakes hyaluronan an effective pro-drug, primarily targeting the carried drug to tumors or the liver, because these tissues have high contents of the cell surface receptor for hyaluronan.        
High molecular weight hyaluronan has been coupled with diethylenetriamine pentaacetic acid (DTPA) (Gouin and Winnik, 2001), where the DTPA groups are used to bind radionuclides for use in cancer therapy, and the hyaluronan targets the complex to tumor cells. Low molecular weight hyaluronan has been coupled to DTPA to chelate Gd+3, and is useful as an imaging contrast agent, targeted to cell surface receptors in tumor tissue. Hyaluronan with attached carborane, targeted to tumor cells, may be employed as an agent in boron neutron capture therapy (DiMeo et al, 2007).
Hyaluronan with covalently attached β-cyclodextrin molecules is able to complex and protect small guest molecules such as ibuprofen (Charlot et al., 2006).
Hyaluronan has been sulfated to form an anticoagulant polymer with reduced platelet attachment (Crescenzi et al, 2002).
Deacetylated hyaluronan, which is a soluble polymer with both cationic (positive) and anionic (negative) groups, bound anionic alginate to form an insoluble polyelectrolyte complex.
Attachment of lipids to hyaluronan (Dan et al., 1998; Schnitzer et al., 2000; Oohira et al., 2000; Ruhela et al., 2006) allows the polysaccharide to be anchored in lipid bilayers, to bind low density lipoproteins, or to affect cell behavior on substrates.
Synthetic polymers and polypeptides can be grafted onto the hyaluronan polymer (or vice versa), providing a variety of elaborate tree-like assemblies, with functionality based on the attached branches. Some of these graft copolymers are suggested for use in DNA delivery.
Hyaluronan derivatized with reactive pendant groups that can become crosslinked following photochemical activation have been prepared for in situ crosslinking. (see chemically crosslinked hyaluronan gels, below).
Insoluble or Slowly Soluble Derivatives of Hyaluronan
Hyaluronan fully esterified by ethyl or benzyl groups is insoluble. This material has proven to be highly biocompatible, and has been manufactured as anti-adhesion fabrics, sponges, membranes, skin grafts, and tissue engineering scaffolds. It is successfully used in general surgical applications, especially abdominal surgery, to minimize post-operative adhesions. It is also used in repair of the tympanic membrane of the ear, and in sinus surgery. The material may also contain fibronectin or other growth factors. Tissue engineering materials may be seeded with cells.
Bioactive materials can also be built on the solid benzyl ester of hyaluronan, coated with two types of multilayers: (poly(dimethyldiallylammonium chloride) and polystyrene sulfonate), followed by poly-D-lysine and an antibody to TGF-β1 (Pastorino et al., 2006).
Hyaluronan modified with a positively charged carbodiimide, mixed with a similarly modified carboxymethyl cellulose, forms a polyelectrolyte complex, in which the positively charged pendant groups and the negatively charged carboxyl groups form electrostatic interactions. This material is successfully used as an anti-adhesion film for surgical applications. It is also used as a coating on polypropylene mesh for surgical applications (see below, for surface attachment applications).
Physical Gels of Hyaluronan
Hyaluronan can form gels by an unknown mechanism, after heating and cooling procedures (Takahashi et al., 2000; Fujiwara et al., 2000). The inter-chain associations, functionally similar to chemical crosslinks, may be aggregates that would be redissolved only over a long time period.
Hydrophobic alkyl groups can be attached to hyaluronan to form amphiphilic polymers that associate strongly to form reversible physical gels (Pelletier et al., 2000; Dausse et al., 2003; Huin-Amargier et al., 2005; Ml{hacek over (c)}ochova et al., 2006; Mrá{hacek over (c)}ek et al., 2007).
Attachment of lactic acid oligomers to a mixed salt (Na+, cetyltrimethylammonium) form of hyaluronan leads to gel formation (Pravata et al., 2008).
Host-guest interactions lead to formation of a gel when hyaluronan having bound β-cyclodextrin is mixed with hyaluronan having a bound acylurea. The pendant groups complex together, linking the polymer chains noncovalently ({hacek over (S)}oltés and Mendichi, 2003; {hacek over (S)}oltés et al., 2004).
Chemically Crosslinked Hyaluronan Gels
Crosslinked gels of hyaluronan have found numerous applications. The gels may be used in the form of sieved particles, spherical microparticles, membranous films, or sponges. In some cases, the gel is employed as a long residence form of the polymer. Thus it is used in preparations for treatment of joint pain and cartilage defects (Balazs et al., 1993; Barbucci et al., 2002a; Balazs, 2004; Asari, 2004; Miller and Avila, 2004), where the turnover of soluble hyaluronan is rapid. Similarly, antiadhesion or wound dressing films formed from crosslinked material remain in place longer, with lifetimes controlled to match the physiological need, such as medium-term prevention of post-surgical adhesions in abdominal or nasal surgery. It can also serve a long term space-filling function in tissue augmentation, for use in vocal fold tissue, urinary sphincter, or facial wrinkles or scars. In other cases, the main function of the gel is to serve as a reservoir of slowly released hyaluronan fragments having angiogenic or chondrogenic effects. Because hyaluronan degradation is aided by reactive oxygen species generated in inflammation, the gels are plausibly termed inflammation-responsive materials.
The hyaluronan crosslinks in the gels can be created using a wide variety of chemical agents such as bisepoxides, divinylsulfone, glutaraldehyde (Crescenzi et al, 2003), carbodiimides, alkyl diamines (Barbucci et al., 2000b, 2000c, 2006), thiols, cystamine (Lee et al., 2007a), photosensitive groups, etc. as reactive species, or via the elegant Ugi and Passerini multicomponent condensations (de Nooy et al, 2000). Alternatively, the hyaluronan may be treated in a manner that results in ester formation between carboxyl groups and alcohol groups on separate hyaluronan chains. The hyaluronan component may be in its native form, or subjected to modifications such as esterification, sulfation (Barbucci et al., 2000b, 2006), oxidation of hydroxymethyl groups with subsequent esterification, attachment of functional amines (Crescenzi et al 2003), or N-deacetylation followed by modifications of the amino group such as sulfation (Crescenzi et al, 2002a). The gel porosity can be altered by treatment with bubbled CO2 (Barbucci and Leone, 2004; Leone et al., 2004).
The porous hyaluronan gels can be loaded with metal ions (Giavaresi et al., 2005), drugs (Barbucci et al., 2005a), proteins, or other therapeutic agents for slow release, resulting from inhibition of the diffusion of the added species. Tissue engineering matrices of hyaluronan gel may be seeded with cells, or crosslinked using the facile click-chemistry in the presence of cells (Crescenzi et al/2007).
Residual activated but incompletely reacted groups can be used to covalently attach drugs or other agents, making the hyaluronan gel a drug carrier. In these cases, the drug release must follow degradation of the hyaluronan or cleavage of the attachment.
Crosslinked matrices of hyaluronan with collagen protein are proposed for tissue engineering (Crescenzi et al 2002b).
In some cases, the mode of hyaluronan gel interaction with added species can be either simple mixture or a covalent attachment. Hyaluronan gels have been proposed to carry cell-adhesive peptides or proteins such as antibodies, growth factors, or thrombin. They may carry enzyme inhibitors such as phospholipase A2 inhibitor or anti-inflammatory vitamin E succinate. They may be used to provide slow release of siRNA to interfere with the synthesis of selected proteins.
Hyaluronan Attached to Surfaces
Hyaluronan-coated articles have been created to improve biocompatibility, especially for surfaces in contact with blood, and to provide specific desirable attributes such as lubricity, reduced nonspecific protein adsorption, and reduced tissue and bacterial cell adhesion (Kito and Matsuda, 1996; Hoekstra, 1999; Barbucci et al. 2003a; Morra, 2005; Taglienti et al, 2006). Specific adhesion of cells expressing hyaluronan receptors can also be achieved, and adhesion of other cell types can be mediated by attachment of cell adhesion-mediating peptides or proteins. The hyaluronan may be physically coated (adsorbed) on the underlying material, or, much more commonly, may be chemically crosslinked to it. The chemical crosslinks may be electrostatic or covalent in nature.
Physical Attachment of Hyaluronan to Surfaces
Hyaluronan has a weak tendency to physically adsorb onto surfaces. Plastic (mostly modified polystyrene) tissue culture and microtiter plates can adsorb hyaluronan from aqueous solutions (especially, 0.1 M sodium bicarbonate) with sufficient stability that the attachments can be exploited in assays for specific binding proteins or cells expressing the CD44 or RHAMM receptors (Delpech et al., 1985; Goetinck et al., 1987; Barton et al., 1996; Catterall et al., 1997; Lokeshwar and Selzer, 2000; Lesley et al., 2002). An important aspect of these weak attachments is the availability of the hyaluronan for specific binding interactions with other species. A drawback of adsorption as an approach to immobilization is the lack of long term stability. Some increase in stability can be obtained if the hyaluronan is dried on the surface (Park and Tsuchiya, 2002). The same effect of drying was observed for hyaluronan adsorbed to silica that had been pretreated with oxygen plasma to add —OH groups (Khademhosseini et al., 2004; Suh et al., 2005; Fukuda et al., 2006). In the latter reports, the molecular weight of the hyaluronan was found to be an important consideration, possibly because high molecular weight chains should have an increased tendency to form interacting networks on the surface during the drying process (Cowman and Matsuoka, 2005). Adsorption of hyaluronan to surfaces can also be exploited to form patterned surfaces, using microcontact printing or molding approaches (Khademiosseini et al., 2004; Fukuda et al., 2006). Adsorption to silica and poly(hydroxyethyl methacrylate) was acceptable, but was poor to polystyrene unless the surface was pre-treated with oxygen plasma (Suh et al., 2004). Patterned hyaluronan on silica resists protein or cell adhesion, whereas bare silica sections will bind fibronectin and cells. Subsequent coating of the hyaluronan sections with polylysine or collagen can be employed to form adhesive surfaces for a second cell type, thus allowing patterned co-cultures to be created (Khademhosseini et al., 2004; Fukuda et al., 2006).
Hyaluronan can be adsorbed on a polyurethane surface that has been previously coated with a gelatin layer. Photo-crosslinking of the hyaluronan layer stabilizes it for use as an anti-thrombotic coating for the luminal surface of a narrow vascular graft (Kito and Matsuda, 1996).
Hyaluronan can be entrapped at a polyethylene surface in a microcomposite structure for use in joint replacement implants. The material is formed by allowing a silyl derivative of hyaluronan to penetrate a porous polyethylene preform, crosslinking it in place, hydrolyzing the silyl groups, adding a surface coat of hyaluronan, crosslinking that layer, and finally compressing the material to collapse the porous structure into a hyaluronan-coated solid with excellent wear properties (Zhang et al., 2006, 2007).
Covalent Attachment of Hyaluronan to Metallic Surfaces
Stable immobilization of hyaluronan on surfaces is achieved by covalent attachment of the polysaccharide, either directly to the surface, or via attachment to a bridging adhesive polymer layer.
Metal substrates (stainless steel, nickel titanium, titanium) are of interest, with respect to medical uses as stents, guide wires, dental and orthopedic implants, sensors, or other devices. The strategy for attachment of hyaluronan is to generate functional groups on the metal surface, and then employ appropriate chemistry to link the hyaluronan molecules.
Functional group generation on metal surfaces can be achieved by methods such as 1) formation of an oxide layer on the metal surface, then reaction with a functional silane derivative (U.S. Pat. No. 5,356,433; U.S. Pat. No. 5,336,518; Pitt et al., 2003); 2) plasma treatment in the presence of air, argon, acetaldehyde, allylamine, hexafluorobutylmethacrylate, etc., to generate groups such as aldehydes and amines on the metal surface (U.S. Pat. No. 5,356,433; U.S. Pat. No. 5,336,518; Thierry et al., 2004; Morra et al., 2006); 3) dip coating with an adhesive acrylic polymer bearing isocyanate groups (U.S. Pat. No. 5,037,677); 4) dip coating with polyethyleneimine having free amine groups (Thierry et al., 2003b); 5) dip coating with dopamine to form an adhesive polymer layer having quinone-like properties (Lee et al., 2007b).
The covalent attachment of hyaluronan to these modified surfaces can be achieved by approaches such as 1) reaction with surface amine groups via carbodiimide activation of hyaluronan carboxyl groups (Larsson, 1987; U.S. Pat. No. 5,356,433; U.S. Pat. No. 5,336,518; Thierry et al., 2004; Morra et al., 2006); 2) reaction of surface amines with the reducing end aldehyde function of short hyaluronan chains to form a Schiff base which is subsequently reduced (U.S. Pat. Nos. 4,613,665 and 4,810,784); 3) reaction of surface aldehyde groups with an adipic dihydrazide derivative of hyaluronan (Pitt et al., 2003); 4) urethane link formation between surface isocyanate groups and hydroxyl groups of hyaluronan (U.S. Pat. No. 5,037,677); 5) attachment of surface quinone-like groups of polydopamine with thiol or amine derivatives of hyaluronan (Lee et al., 2007a).
The hyaluronan-derivatized metal surfaces can differ in the degree to which the hyaluronan can bind proteins, cells, etc. The surfaces are generally intended to be resistant to nonspecific adsorption of proteins or cells. There is special interest in surfaces that do not bind fibrinogen or platelets. Cells expressing CD44 can adhere to some surfaces. There is currently insufficient knowledge about the best approaches to control the frequency of hyaluronan attachments to the surface, and thus control the degree to which the surface-bound hyaluronan is available to show specific binding interactions with proteins or cells.
One special metallic surface that is easily and specifically reacted for attachment of hyaluronan is gold. Gold nanoparticles were reacted with hyaluronan bearing thiol groups, previously attached via carbodiimide activation of carboxyl groups and coupling to cystamine, followed by reduction of the pendant disulfide to a thiol (Lee et al., 2006). Many small nanoparticles could be linked to a single long polymer of hyaluronan, resulting in a necklace of gold nanoparticles.
Covalent Attachment of Hyaluronan to Polymeric Surfaces
Attachment of hyaluronan to polymeric substrates has also been widely investigated for use in implants, catheters, etc. Among the polymeric substrates investigated for hyaluronan attachment are polystyrene, poly(methyl methacrylate), silicone rubber, poly(tetrafluoroethylene) [Teflon], poly(ethylene terephthalate), polyurethane, poly(vinyl alcohol), polyethylene, and polypropylene. There are patents dating from the mid 80's on hyaluronan-modified polymers (e.g., Balazs, Leschiner, and coworkers patents 1984, 1985; Beavers and Halpern patents 1987, 1989, 1991; Larm patents 1986, 1989). There are a number of strategies used to form stable covalent attachments of hyaluronan to surfaces.
The polymer surface may be treated with a plasma to provide appropriate functional groups such as amines. For example, commercially available aminated polystyrene materials may be reacted with carbodiimide activated hyaluronan (Frost and Stern, 1997) or reacted first with an azide-bearing group then photocrosslinked to hyaluronan (Joester et al., 2006). Ammonia plasma treatment can be used to aminate the surface prior to photo-immobilization of an azidophenyl hyaluronan derivative (Barbucci et al., 2005b). Ammonia plasma treatment followed by reaction with succinic anhydride produces surface carboxyl groups that can be reacted with an adipic hydrazide derivative of hyaluronan (Mason et al., 2000). The surface can be activated by air plasma treatment, reaction with polyethyleneimine, and subsequently reacted with carbodiimide-activated hyaluronan (Morra and Cassinelli, 1999; Cassinelli et al., 2000). The substrate may be activated by plasma polymerization of acetaldehyde, then Schiff base formation with poly(allylamine), followed by reaction with carbodiimide-activated hyaluronan (Thierry et al., 2004).
Poly(ethylene terephthalate) can be oxidized in base at high temperature to form carboxyl groups, then coated with a cationic polymer prior to hyaluronan (Liu et al., 2006).
Hyaluronan can be photochemically immobilized on silicone rubber, after initial immobilization of a polyacrylamide layer, then a hyaluronan layer, both linked covalently using the 4-benzoyl benzoic acid derivatives (DeFife et al., 1999).
Hyaluronan can also be covalently attached to more adhesive polymer layers that are strongly attached to the substrate. Coating PMMA with an acrylic polymer bearing isocyanates can be used to attach hyaluronan via urethane linkages to its hydroxyl groups (Lowry and Beavers, 1994; U.S. Pat. Nos. 4,663,233; 4,801,475; and 5,037,677). Dopamine coating on polymeric surfaces leads to a polymeric coating containing quinone-like groups to which thiol or amine-derivatized hyaluronan can attach (Lee et al., 2007b).
As for the attachment of hyaluronan to metallic surfaces, the frequency of attachment points of the hyaluronan molecule to polymeric surfaces can affect its ability to interact with proteins or cells. When attachment points are closely spaced, the hyaluronan can be resistant to degradation by hyaluronidase, and thus show long term stability (Lowry and Beavers, 1994). In most published studies, hyaluronan-coated polymer materials are reported to be anti-adhesive to most proteins and cells, but show specific adhesion of CD44+ cells.
Covalent Attachment of Hyaluronan to Silica Surfaces
Hyaluronan can be covalently attached to glass (silica). Applications of the coated silica are primarily in cell culture. Many silica surfaces modified with hyaluronan are notable for inhibiting cell adhesion.
Most of the silica-based surfaces are derivatized via silane chemistry. If the silica is reacted with an aminosilane which becomes covalently attached as a monolayer presenting amino groups, then hyaluronan that has an azidophenyl group can be photo-immobilized and even micropatterned on the surface using a photolithographic mask procedure (Barbucci et al., 2003; Chiumiento et al., 2007). A surface bearing aminosilanes can also be reacted with carboxyl groups of hyaluronan via carbodiimide coupling (Albersdörfer and Sackmann, 1999; Ibrahim et al., 2007; Joddar et al., 2007). Fluoroalkylsilanes on silica can be photo-crosslinked with hyaluronan bearing azidoaryl groups (Wang et al., 2006). Chlorotrimethylsilane on silica provides a hydrophobic surface that can adhere to poly(lactic-co-glycolic acid), allowing subsequent coating with polyethyleneimine and finally attachment of hyaluronan by carbodiimide chemistry (Croll et al., 2006). It is also possible to make a silane-bearing derivative of hyaluronan, which can directly react with the bare silica surface (Pasqui et al., 2007). This latter method may be preferred because fewer of the hyaluronan carboxyl groups are derivatized, and therefore remain able to participate in hyaluronan interactions with other species such as proteins. Hyaluronan attached to silica by these procedures does not promote cell adhesion (Barbucci et al. 2003b; 2005b; Pasqui et al., 2007; Chiumiento et al., 2007).
An alternative approach to silica surface modification is the use of an ethylene plasma to create a hydrophobic surface, oxidation of that layer in an air plasma, followed by coating with polyethyleneimine. The amine groups can then be reacted with hyaluronan carboxyl groups via carbodiimide chemistry (Morra et al., 2003).
Covalent Attachment of Hyaluronan to Quantum Dots
Quantum dots of CdSe/ZnS, with attached ligands containing terminal carboxyl groups, have been derivatized by carbodiimide-mediated reaction with amine groups on an adipic acid dihydrazide derivative of hyaluronan (Kim et al., 2008).
Electrostatic Attachment of Hyaluronan to Surfaces
Electrostatic immobilization of hyaluronan has also been widely investigated. A metal or silica surface may be precoated with cationic polymers such as polyethyleneimine, chitosan, or polylysine prior to electrostatic attachment of hyaluronan (Morra et al., 2003; Burke and Barrett, 2003; Thierry et al. 2003b; Hahn and Hoffman, 2005; Tezcaner et al., 2006). Gold-coated silica substrate can be reacted with carboxylated alkylthiols to form an anionic monolayer, coated with cationic polyethyleneimine, and then electrostatically linked to hyaluronan (Kujawa et al., 2005). Nanospheres of poly-ε-caprolactone can be formed in the presence of a cationic surfactant such as benzalkonium chloride, and then coated with an electrostatically bound hyaluronan layer (Barbault-Foucher et al., 2002).
Several research groups have worked extensively on the formation of coatings that are formed by laying down alternating electrostatically-bound layers of hyaluronan and a cationic polymer. The coating is sometimes called a polyelectrolyte multilayer. The technique is frequently referred to as layer-by-layer assembly. The layers are not perfectly smooth, as the polymers have a tendency to form islands of adsorbed material, and thus there can be extensive interpenetration of the anionic and cationic polymer layers. The coatings can be used to carry bioactive agents (see below).
One research group has extensively investigated polyelectrolyte multilayers composed of hyaluronan and either polylysine or chitosan, usually deposited directly on glass or quartz substrates (Picart et al., 2001, 2002, 2005; Richert et al., 2004a, 2004b, 2004c, 2006; Collin et al., 2004; Zhang et al., 2005; Etienne et al., 2005; Schneider et al., 2006, 2007a, 2007b; Francius et al., 2006; Tezcaner et al., 2006). Using a robotic coating device, the assemblies could be built with many (ca. 20-100) layers. In some studies, the coatings were crosslinked after deposition. The stiffness parameters of the coatings were investigated by nanoindentation of a colloidal probe tip in an atomic force microscope, or by piezo-rheometer. It was observed that crosslinking increased stiffness and simultaneously increased cell adhesion. The crosslinked films were also resistant to enzymatic degradation. The crosslinked films were proposed to be useful in delivery of drugs such as paclitaxel or diclofenac (see below). Most recently, assemblies of hyaluronan with collagen protein, and hyaluronan with an aminated derivative of hyaluronan were prepared and characterized.
Another research group has extensively studied polyelectrolyte multilayers formed with hyaluronan and chitosan (Thierry et al., 2003a, 2003b, 2004, 2005; Kujawa et al., 2005, 2007). The coatings were immobilized on several different substrates, including polyethyleneimine on metal; polyethyleneimine on a carboxyalkylthiol-derivatized gold on silica; quartz, and artery walls.
Additional groups have provided other very interesting and creative studies of polyelectrolyte multilayers containing hyaluronan. Liu et al. (2006) prepared a hyaluronan-chitosan multilayer film for use in coating poly(ethylene terephthalate) in a microfluidic device. Veerabadran et al. (2007) built a hyaluronan-polylysine multilayer coating that was able to encapsulate and protect stem cells. Lee at al. (2007b) devised a spherical microshell composed of a crosslinked hyaluronan-polylysine multilayer, formed initially around a disulfide-crosslinked hyaluronan hydrogel microsphere that was liquified by reduction and allowed to diffuse out. The redox properties of polyelectrolyte multilayers composed of hyaluronan and the globular protein myoglobin have been studied as models for novel coatings for biosensors, bioreactors, and other biomedical devices (Liu and Hu, 2006; Lu and Hu, 2007).
Hyaluronan Attached to Supported Lipid Bilayers or Liposomes
Supported lipid bilayers can be used to immobilize hyaluronan. Sengupta et al. (2003) used a supported bilayer, deposited on glass, and formed with a small fraction of lipid containing a nickel-chelating head group. Using histidine-tagged p32 protein that binds hyaluronan, the polysaccharide was then bound noncovalently to the surface. Benz et al. (2004) used a lipid bilayer on mica to attach hyaluronan in several ways. A lipid carrying a biotin group allowed a layer of streptavidin to be bound to the bilayer, and then biotin-labeled hyaluronan could be noncovalently bound to the surface. They also bound hyaluronan covalently to an amine-bearing lipid, using carbodiimide activation of hyaluronan carboxyl groups. The bound hyaluronan layer could be subsequently crosslinked to form a stable coating. Richter et al. (2007) used the biotin-streptavidin-biotin technique to attach hyaluronan to a lipid bilayer, but using hyaluronan end-labeled with biotin gave a better result than the approach of Benz et al. (2004).
Margalit and coworkers pioneered the use of liposomes with bound hyaluronan. Attachment of hyaluronan via carbodiimide activation of carboxyl groups and reaction with amine-bearing lipids resulted in stable hyaluronan-coated lipid vesicles (U.S. Pat. No. 5,401,511; Yerushalmi et al., 1994; Yerushalmi and Margalit, 1998; Peer and Margalit, 2000). The interior of the lipid vesicles could be loaded with growth factors for use in wound healing, or any of a number of drugs. The hyaluronan coat provided adhesion to certain types of cells expressing CD44 receptors, for extended residence time at the wound site. The hyaluronan coat also provided protection of the liposome during lyophilization and reconstitution procedures that allow long term storage of liposome-based therapeutics (Peer et al., 2003). Szoka and coworkers (Eliaz and Szoka, 2001; Eliaz et al., 2004a, 2004b) produced liposomes with hyaluronan oligosaccharides bound to amine-bearing lipids via reductive amination. These liposomes could carry the chemotherapeutic agent doxorubicin, and target it to tumor cells overexpressing the CD44 receptor. The hyaluronan-coated liposomes provided higher potency in tumor cell cytotoxicity, and lower toxicity to other cell types. Peer and Margalit (2004a, 2004b) further established the utility of high molecular weight hyaluronan as a targeting agent for liposomes carrying anti-tumor agents (doxorubicin, mitomycin C). Another use of hyaluronan-coated liposomes, proposed by Margalit and coworkers (Fischer et al., 2005), was as a carrier of an enzyme to protect against nerve toxin organophosphates. It should be noted that the hyaluronan coating in all of these liposome studies was not involved in binding or sequestering the bioactive agents, which were encapsulated within the liposomes.
Surface-Immobilized Hyaluronan as a Carrier of Bioactive Agents
Layer-by-layer assemblies of hyaluronan and chitosan or hyaluronan and polylysine have been suggested as carriers of bioactive agents such as arginine (Thierry et al., 2003a), sodium nitroprusside (Thierry et al., 2003b), paclitaxel (Schneider et al., 2007b) and sodium diclofenac (Schneider et al., 2007b). The enzyme trypsin, when immobilized on a chitosan layer of a hyaluronan-chitosan multilayer film, was more active in protein digestion than free trypsin (Liu et al., 2006). Basic fibroblast growth factor (bFGF), adsorbed onto a polylysine layer of a hyaluronan-polylysine film, was more effective than free bFGF in aiding adhesion and maintaining differentiation of photoreceptor cells (Tezcaner et al., 2006). Covalent attachment of gelatin to a hyaluronan-chitosan film was used to aid fibroblast adhesion (Croll et al., 2006).
Chemically modified hyaluronan can also be incorporated into a surface coating. DTPA-modified hyaluronan in a coating has been suggested as a carrier of radionuclides to inhibit cell proliferation on stents (Thierry et al., 2004). Hyaluronan can be derivatized with the RGD peptide that mediates cell adhesion, to facilitate integration of an implanted material with surrounding tissue, esp. bone (Pitt et al., 2003). Hyaluronan chemically derivatized with the drug paclitaxel was used with chitosan to form a multilayer (Thierry et al., 2005). Sulfated hyaluronan, with nearly 90% of the hydroxyl groups sulfated, has also been extensively studied in attachment to surfaces. The sulfated hyaluronan is so significantly modified that its properties are not similar to hyaluronan, but it has excellent anticoagulant activity. It has a variable affinity for cell adhesion, generally being more adhesive than hyaluronan. In complexation with Cu+2 ions, it is angiogenic. Barbucci has published a number of reports concerning sulfated hyaluronan on surfaces. (Chen et al., 1997; Magnani et al., 2000, 2004; Barbucci et al., 2000c, 2002b, 2002c, 2003b, 2005c, 2005d; Hamilton et al., 2005; Chiumiento et al., 2007)
Hyaluronan attached to silica has been reported to have altered protein or cell surface interactions relative to free hyaluronan. The altered interactions can be due to the chemical changes in the polymer caused by the procedures used to attach hyaluronan to a surface (e.g., effective loss of carboxyl groups). An example is the binding with apparent conformation change of the protein fibronectin on a surface containing photoimmobilized hyaluronan, where the hyaluronan has lost carboxyl function and has an extreme degree of attachment frequency (Barbucci et al., 2005c). Similarly, fibrinogen adsorbed onto a photoimmobilized hyaluronan surface cannot bind platelets, but fibrinogen can do so if independently covalently attached to the surface (Chiumiento et al., 2007). Serum proteins can adsorb to a surface having photoimmobilized hyaluronan, and some bind strongly enough that a combination of detergent, urea and dithioerythritol is required to release them (Magnani et al., 2004).
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